Abstract Spinal muscular atrophy (SMA) is caused by reduced levels of survival motor neuron (SMN) protein. The disease manifests by loss of alpha motor neurons in the spinal cord with symptoms of severe muscle weakness, atrophy, disability, and in many cases leads to death. The incidence of SMA is approximately 1 in 10,000 live births and is the leading genetic cause of infant mortality. Currently there is no effective treatment for the disease. Gene therapeutic and antisense-oligonucleotide studies have shown successful elevation of SMN protein level and consequent protection from motor neuron loss if given within a therapeutic window. Emerging results from clinical trials also show that SMN protein level and motor function can be significantly improved, but only if intervened early. The therapy shows modest functional improvement or no effect if there was significant prior loss of motor neurons. This leaves a major therapeutic gap for the predominant number of SMA patients, and critically requires additional methods of intervention. Such patients are characterized by chronic muscle weakness and fatigue due to reduced neural input from motor neuron loss. Our preliminary murine studies show inclusion of skeletal-muscle activators, which increase output of muscle force and enhance endurance in short-term studies has the potential to be beneficial for all who suffer from SMA. Enhanced muscle force can be achieved by sensitizing the sarcomere to calcium. The fast-skeletal-muscle troponin activator tirasemtiv is an example of a small molecule that acts in this manner and increases muscle force response with sub-maximal nerve stimulation. In our studies, a single dose of tirasemtiv was effective in enhancing muscle force output in SMA mice, thus establishing the premise for our studies. Troponin C (TnC) slows the rate of calcium release from the troponin complex and sensitizes the sarcomere to calcium. We have engineered multiple TnC variants that can modulate Ca2+ sensitivity of force development by over an order of magnitude. With robust preliminary results, we propose to further our investigations and study the long-term effects of targeting myofilament calcium sensitivity as a means to increase muscle force production. This will allow us to gain insight on the effect that a sustained calcium sensitizing activator has on muscle function, endurance and potential feedback on the entire SMA motor unit. To achieve these goals in Aim 1 we will use AAV9-gene delivery to overexpress a calcium-sensitized TnC (L48Q) to enhance force either before or after motor neuron loss by delivery to neonates or adults, respectively. In Aim 2, based on novel preliminary data, we will enhance muscle relaxation by delivery of parvalbumin to subsidize muscle with its natural relaxation factor, in conjunction with the TnC. Collectively these studies will provide important insight into the long-term effects of calcium sensitivity and buffering as a means to enhance muscle force production in SMA and more broadly other conditions of muscle weakness.